Creatine kinase is an essential enzyme in the maintenance and regulation of intracellular ATP levels. It catalyzes a phosphoryl transfer reaction in which creatine becomes reversibly phosphorylated. Phosphocreatine acts as a reservoir of high energy phosphate for the cell to maintain ATP homeostasis. There are 3 isozymes of creatine kinase (CK), muscle, brain and mitochondrial. Much of my current research focuses on the brain isozyme of CK. The brain isozyme of creatine kinase (CK-BB) is a key enzyme in the regulation of cellular energetics. CK-BB has also been associated with several disease states, including several types of cancer. In contrast to the muscle and mitochondrial isozymes of CK, CK-BB is regulated by steroid hormones, phospholipase C and protein kinase C. Brain CK is currently being explored as a drug target for chemotherapy. In collaboration with the Stroud lab, initial crystallization trials have yielded high quality 1005m crystals for human brain CK and have formed under a set of unique conditions not previously found for CK. These crystallization conditions would favor heavy atom replacement, which has been impossible with the rabbit muscle CK crystals due to instability. Since a high quality native data set for rabbit muscle CK has been collected by the Stroud lab, the structure of CK could be solved using the brain isozyme apoenzyme crystals soaked with a heavy atom, such as the cysteine modifying reagent PCMB. A necessary step in deducing the structure of a protein is having the confirmed primary sequence. In collaboration with Dr. Fred McLafferty at Cornell University, we have confirmed the primary sequence of both human muscle and brain. CK isozymes using high resolution Fourier transform tandem mass spectrometry. In order to resolve sequence discrepancies in the literature, this work relied heavily on sequence alignments generated using CGL computers and GCG software. The details of this work were published in the Proceedings of the National Academy of Sciences USA in December 1995. I am currently attempting to identify the phosphorylation site(s) of the brain isozyme of CK isolated from human placenta. Knowledge of the phosphorylation site will provide information about brain CKUs mode of interaction with protein kinase C. Based on structural and sequence data available for PKC and PKC substrates, inhibitors can be designed using the information.